This invention relates to the drug potential of anticonvulsants in the treatment of stroke, particularly, several xcex942-1,2,3-triazoline and aminoalkylpyridine (AAP) anticonvulsants that seem to work by impairing the excitatory amino acid (EAA) L-glutamate (L-Glu) neurotransmission, as antiischemic agents, useful in the treatment of stroke victims.
There is strong evidence that the xe2x80x9cexcitotoxicxe2x80x9d action resulting from the excessive accumulation of L-Glu plays a prominent role in human epilepsy as well as brain ischemia/stroke, leading to neuronal dysfunction and cell death. The 1,2,3-triazolines and the aminoalkylpyridine (AAP) metabolite analogues are two groups of novel anticonvulsants discovered in the Applicant""s laboratories. These are very effective in the kindling and in the maximal electroshock (MES) seizure models of epilepsy, the best analogies to human partial seizures, where EAA neurotransmission plays an important role. Thus it is logical to expect that the anticonvulsant triazolines and AAP metabolite analogues would evince beneficial therapeutic potential in cerebral ischemia.
The ability of the triazolines and AAP compounds to afford protection and reduce neuronal degeneration are assessed in animal models of stroke, by utilizing the bilateral carotid occlusion model in the gerbil and the middle cerebral artery occlusion (MCAO) model in the rat. Post-ischemic gerbils undergo a predictable pattern of behavioral changes and the effects of drugs in producing alterations in this pattern are monitored by determining the post ischemic changes in locomotor activity as well as by changes in radial arm maze performance, and corroborated by post reperfusion histopathological assessment. In the MCAO rat model, a focal stroke model, drug effects are evaluated from their ability to reduce the infarct volume following MCAO.
There is a desperate need for clinically effective chemotherapeutic agents for intervention in and management of cerebral ischemia resulting from stroke. In the U.S. alone, 1.1 million individuals suffer stroke annually; it is the most common, and devastating neurological condition that kills more than a quarter million Americans every year and the leading cause of long-term intellectual and physical disability. In the past decade, it has become increasingly evident from data from numerous laboratories that EAA neurotransmission plays an important role in ischemic brain injury occurring in stroke and other neurological disorders (McCulloch, J., et al., Ed., xe2x80x9cFrontiers in Pharmacology and Therapeutics: Excitatory Amino Acid Antagonists.xe2x80x9d, Oxford, UK; Blackwell Scientific Publishers, 287-326, 1991: Choi, D. W. and Rothman, S. M., Annu. Revs., Neurosci., 13, 171-182, 1991; Takagi, K., et al., J. Cereb. Blood Flow Metab., 13, 575-585, 1993; Graham, S. H., et al., J. Cereb. Blood Flow Metab., 13, 88-97, 1993; Muir, K. W., and Lees, K. R., Stroke, 26, 503-515, 1995). The excessive accumulation of the excitatory neurotransmitter L-Glu, followed by its excitotoxic action, has been strongly implicated in the cascade of pathological mechanisms that cause neuronal dysfunction and cell death in cerebral hypoxia-ischemia resulting from stroke, cardiac arrest, or mechanical brain injury. Thus, the EAA neurotransmitter systems may be considered potential therapeutic targets and development of agents that are EAA antagonists may constitute novel and effective therapies, as cytoprotective agents, in stroke.
It is accordingly one object of the present invention to provide novel xcex942-1,2,3-triazolines and AAP compounds and their method of preparation.
It is a further object of the present invention to provide antiischemic/antistroke agents which comprise triazolines and AAP compounds
A further object of the present invention is to provide a method for the treatment of cerebral ischemia resulting from stroke, by administration of an effective amount of the triazoline and AAP compounds of this invention.
A further object of the present invention is to provide triazolines and AAPs bearing three different pyridyl substituents and a pyrrolidinone group, and methods for their use in the treatment of neurological disorders such as cerebral ischemia resulting from stroke and also in the treatment of epilepsy.
A still further object of the present invention is to provide triazolines and AAP compounds, as inhibitors of the EAA neurotransmitter L-glutamate. The triazolines and AAPs of this invention afford pronounced protection in the maximal electroshock seizure (MES) model in both mice and rats, by the intraperitoneal, intravenous, and oral route, which is indicative of their action as glutamate antagonists.
A still further object of the present invention is to provide antiischemic compositions that contain as the essential ingredient certain triazolines and AAPs and that are highly effective by the intraperitoneal and intravenous routes, the preferred routes of administration, in stroke victims, and use of these triazolines and AAPs as effective antiischemic drugs in the treatment of cerebral ischemia resulting from stroke.
Other objects and advantages of the present invention include use of the triazolines and AAPs in the treatment of stroke and epilepsy and also other neurological disorders such as Parkinson""s disease, by virtue of their action as EAA antagonists and inhibitors of L-glutamate neurotransmission.
In satisfaction of the foregoing objects and advantages, there are provided by this invention several triazolines and AAPs which are useful as antiischemic/antistroke drugs. The various groups of triazolines and AAPs substituted with the various pyridyl groups and also the pyrrolidinyl group, may be characterized by the following general formulae: 
wherein R1 is 3,4- or 3,5-dichloro, p- or m-chloro, p- or m-bromo, p- or m-fluoro, p- or m-trifluoromethyl, p- or m-lower alkyl, p- or m-lower alkoxy or hydrogen.
Also provided by this invention are non-toxic antiischemic compositions that are intraperitoneally and intravenously active and comprise as the active ingredient, a compound selected from those of the formulae (I-VII), wherein R1 is 3,4- or 3,5-dichloro, p- or m-chloro, p- or m-bromo, p- or m-fluoro, p- or m-trifluoromethyl, p- or m-lower alkyl, p- or m-lower alkoxy or hydrogen.
Also provided are methods for the administration of the antiischemic compositions of this invention to mammals, including animals and humans, in the treatment of cerebral ischemia resulting from stroke, including both global ischemia and focal ischemia.
As indicated above, this invention relates to several groups of compounds belonging to the seven structures (I-VII) shown above, which are useful as antiischemic drugs in the treatment of cerebral ischemia resulting from stroke. In one group of triazolines (I) and AAPs (V), a 4-pyridyl substituent is present, in a second group of these compounds (II and VI), a 3-pyridyl substituent and in a third group (III and VII), a 2-pyridyl substituent is present. Also, in a fourth group of triazolines (IV), a 2-oxo-1-pyrrolidino group is present. In all three groups of AAP compounds, the R2 group is methyl, ethyl or phenyl. The triazolines and AAPs of this invention are further substituted on the phenyl rings by 3,4- or 3,5-dichloro, p- or m-chloro, p- or m-bromo, p- or m-fluoro, p- or m-trifluoromethyl, p- or m-lower alkyl, p- or m-lower alkoxy or hydrogen. The triazolines and AAPs of this invention have potent antiischemic activity and protect the brain from neuronal damage in both global and focal ischemia, and are useful as antiischemic/antistroke drugs in the treatment of cerebral ischemia resulting from stroke in humans.
In one aspect of the present invention, three groups of triazolines and two groups of AAPs, are provided which have potent antiischemic activity and which have the general formulae represented by structures I, II and IV, and V and VI, respectively. In the above formulae, in structures I and II, the 5-substituent is 4-pyridyl or 3-pyridyl and R1 is 3,4- or 3,5-dichloro, p- or m-chloro, p- or m-bromo, p- or m-fluoro, p- or m-trifluoromethyl, p- or m-lower alkyl, p- or m-lower alkoxy or hydrogen. In structure IV, the 5-substituent is a 2-oxo-1-pyrrolidino group and R1 is 3,4- or 3,5-dichloro, p- or m-chloro, p- or m-bromo, p- or m-fluoro, p- or m-trifluoromethyl, p- or m-lower alkyl, p- or m-lower alkoxy or hydrogen. Structures V and VI, are 4-pyridyl and 3-pyridyl AAPs respectively, where R4 is methyl, ethyl or phenyl and R1 is 3,4- or 3,5-dichloro, p- or m-chloro, p- or m-bromo, p- or m-fluoro, p- or m-trifluoromethyl, p- or m-lower alkyl, p- or m-lower alkoxy, or hydrogen. Several of these triazolines and AAP compounds are potent anticonvulsants and are already under U.S. patent protection (P. K. Kadaba, U.S. Pat. No. 4,511,572, 1985; U.S. Pat. No. 4,689,334, 1987; U.S. Pat. No. 4,820,721, 1989; U.S. Pat. No. 5,648,369, 1997; U.S. Pat. No. 6,083,964, 2000).
In a second aspect of this invention, there are provided novel antiischemic compositions which are effective by the intraperitoneal and intravenous routes and are non-toxic, and which comprise as the active ingredient an effective amount of a compound selected from those of the seven groups represented by structures (I-VII), and having 4- or 3-pyridyl, or 2-oxo-1-pyrrolidinyl substituent groups, and R4 and R1 are as described above.
There are further provided by this invention, methods for the administration of the antiischemic compositions to mammals including animals and humans.
In a third aspect of this invention, there are provided triazoline and AAP compounds of the formulae represented by the structures (I-VII), and which exhibit pronounced and selective activity in the MES test and the kindling model of epilepsy, and are useful in the treatment of stroke.
Significance of Pronounced Selective Activity in the MES Test:
Both the triazolines and the AAP compounds of this invention exhibit pronounced and selective anticonvulsant activity in the maximal electroshock seizure (MES) test. While the triazolines show activity in the subcutaneous Metrazole (scMet) test also, the AAPs show hardly any activity in the scMet test. The activity of the compounds of this invention in the MES test is of great significance, because partial seizures in humans correlate positively with experimental seizures elicited by the MES test [Porter, R. J. and Pitlick, W. H., In xe2x80x9cBasic and Clinical Pharmacologyxe2x80x9d, 4th Edn., B. G. Katzung Ed., Appleton and Lange, C. A., 1989, pp 287-303]. Since antiepileptic drugs effective against MES seizures alter ionic transport across excitable membranes, the triazolines and the AAPs that evince significant activity in the MES test, may be expected to attenuate EAA neurotransmission. There is strong evidence that the excitatory neurotransmitter glutamate plays a key role in EAA neurotransmission along limbic circuits which are particularly relevant to kindling epileptogenesis. Since the triazolines and the AAPs are quite effective in the kindling model, both these classes of compounds could be expected to be effective glutamate antagonists.
Previous studies in our laboratories had led to the emergence of the 1,2,3-triazoline heterocycles represented by structures I-IV, as a new class of anticonvulsant agents with a unique mechanism of action quite different from the more traditional anticonvulsants (Kadaba, P. K., J. Med. Chem., 31, 196-302, 1988; xe2x80x9cDrugs of the Futurexe2x80x9d, 15, 1013-1024, 1990; Kadaba, P. K., and Slevin, J. T., Epilepsia, 29, 330, 1988; Kadaba, P. K., and Slevin, J. T., Pharmaceut. Res., 6, S-42, 1989; Kadaba, P. K. and Slevin, J. T., 200th Nat. Meeting of the ACS, Washington, D.C., Abstracts, 56, MEDI 31, 1990; Kadaba, P. K., Stevenson, P. J., Nnane, I. P., and Damani, L. A., Bioorg. Med. Chem., 4, 165-178, 1996). The triazolines afford a high degree of protection in seizure provocation by chemical (scMet) and electrical (MES) stimuli and have good protective indices. They offer complete protection against N-methyl D-aspartate (NMDA)-induced seizures in the mouse at significantly low ED50 values: (Kadaba, P. K., et al., Bioorg. Med. Chem., 4, 165-178, 1996). They show good response on oral administration and a good margin of safety. They compare very well with prototype antiepileptic drugs in both mice and rats. Unlike the prototype drugs, one triazoline represented by structure I, R1=pxe2x88x92Cl, offers complete protection against stimulus-induced electrographic after-discharge seizures and generalized convulsions, in both amygdala-kindled (ED50=215xc2x161 mg/kg) and entorhinal-kindled rats (ED50=423xc2x145 mg/kg), in non-sedative, non-neurotoxic doses (Kadaba, P. K., xe2x80x9cDrugs of the Futurexe2x80x9d, 15, 1013-1024, 1990: Kadaba, P. K. and Slevin, J. T., Pharmaceut. Res., 6, S-42, 1989).
Studies on the metabolism and in vivo and in vitro pharmacology of triazolines represented by structure I and potential metabolites, seem to indicate that the triazolines may be functioning as prodrugs and act by a unique xe2x80x9cdual-actionxe2x80x9d mechanism; while the parent triazoline inhibits the presynaptic release of glutamate (58% at 50 xcexcM and 83% at 100 xcexcM drug concentration), the active B-amino alcohol metabolite displaces  greater than 90% of the binding of [3H]-Glu from glutamate receptors, and 56% of the binding of [3H]MK-801, from the MK-801 sites on the NMDA receptor ionophore complex (Kadaba, P. K., ACS Abstrs. MEDI 144, 1991; Kadaba, P. K. and Slevin, J. T., Epilepsia, 29, 330, 1988; Pharmaceut. Res., 6, S-42, 1989; ACS Abstrs., 56, MEDI 31, 1990; Kadaba, P. K., et al., Bioorg. Med. Chem. 4, 165-178, 1996). Furthermore, radioligand binding studies at ion-channel binding sites using [3H]TBOB, indicates significant activity at Clxe2x88x92 channels ranging from 50 to 63% at 10 xcexcM concentration, for triazolines belonging to structure I (Kadaba, P. K., et al., Bioorg. Med. Chem. 4, 165-178, 1996). Augmentation in Clxe2x88x92 influx is a useful membrane action that reduces membrane excitability or alters circuit behavior to favor inhibition, and thus might help suppress the firing of glutamatergic neurons and hence glutamate release. Such drugs may be most beneficial in the control of prolonged seizures such as in status epilepticus where excessive neuronal firing occurs (Choi, D. W., Cerebrovasc. Brain Metab. Revs., 2, 105-147, 1990). And indeed, the complete protection afforded by triazoline I (R1=p-Cl) against amygdala- and entorhinal-kindled seizures as well as NMDA-induced convulsions is significant, in view of the current concepts regarding the central role of EAA neurotransmission, particularly L-Glu, in the kindling model of human partial epilepsy.
Studies by the Applicant on the metabolism and pharmacology of the triazoline anticonvulsants have led to the evolution and discovery of the aminoalkylpyridines (AAPs) as a unique class of orally active anticonvulsant agents, superior to the triazolines themselves (Kadaba, P. K., et al., Bioorg. Med. Chem., 2, 165-178, 1996; Kadaba, P. K., U.S. Pat. No. 4,511,572, 1985; U.S. Pat. No. 4,618,681, 1986; U.S. Pat. No. 4,689,334, 1987; U.S. Pat. No. 4,820,721, 1981). Work on the aminoalkylpyridines indicate they are non-toxic, and highly effective by the oral route, with protective indices greater than 20. The AAPs also show high anticonvulsant activity in the MES test and are practically inactive in the scMet test (Deshmukh, T. R. and Kadaba, P. K., Med. Chem. Res., 3, 223-232, 1993; U.S. Pat. No. 5,648,369,1997).
Radioligand binding and release studies indicate that the ability of triazolines to impair presynaptic release of glutamate is retained to the full extent or better in the corresponding AAP compounds (V, R1=p-Cl) (74% at 50 xcexcM and 80% at 100 xcexcM drug concentration as also the postsynaptic activity of the xcex2-amino alcohol, albeit at a different site; the AAP compounds weakly displace [3H]DTG, a o specific ligand, with Ki values in the xcexcM range and show no affinity for the PCP sites (Kadaba, P. K., ACS Abstrs. MEDI 073, 1992; Pharmaceut. Res., MNPC 5013, 11, S-120, 1994a; Epilepsia, AES, Dec. 5, 1994b; Deshmukh, T. R., and Kadaba, P. K., J. Pharm. Res. 9, S-109, 1992; Med. Chem. Res. 3, 323, 1993; Kadaba, P. K., and Deshmukh, T. R., ACS Abstrs., MEDI, 1069, 1993a; Amino Acios, June, 1993b; Kadaba, P. K., et al., Bioorg. Med. Chem., 2, 165-178, 1996). As o and PCP sites are two distinct molecular entities (Kamenka, J. M. and Domino, E. F., (Eds), xe2x80x9cMultiple Sigma and PCP Receptor Ligands: Mechanisms for Neuromodulation and Neuroprotection?xe2x80x9d, NPP Books, P.O. Box 1491, Ann Arbor, Mich., 48106, 1992) and the o receptor is not a component of the NMDA receptor-ionophore complex, the potent anticonvulsant activity of the AAPs seems to result from their selective low-affinity interaction at o1 sites. The selectivity of the AAPs for the o receptor sites with no activity at the PCP sites, might also account for the absence of undesirable toxic side effects in these compounds.
The role of EAAs and the NMDA receptor in health and disease are extensively reviewed (Cavalheiro, E. A., Lehmann, J., and Turski, L., Eds., xe2x80x9cFrontiers in Excitatory Amino Acid Researchxe2x80x9d, A. R. Liss, New York, N.Y., 1988; Cotman, C. W., Bridges, R. J., Taube, J. S., Clark, A. S., Geddes, J. W., and Monaghan, D. T., J.NIH Res., 1, 65, 1989; Dingledine, R., Boland, L. M., Chamberlin, N. L., Kawasaki, K., Kleckner, N. W., Traynelis, S. F., and Verdoom, T. A., CRC Crit. Rev. Neurobiol., 4, 1, 1988; Honore, T., Med Res. Rev. 9, 1, 1989; Johnson, G., Ann. Rep. Med. Chem., 24, 41, 1989). Overstimulation of the NMDA receptor by high levels of glutamate has been implicated in both epilepsy (Cavalheiro, E. A., Lehmann, J., and Turski, L., Eds., xe2x80x9cFrontiers in Excitatory Amino Acid Researchxe2x80x9d, A. R. Liss, New York, N.Y., 1988; Fisher, R. S. and Coyle, J. T., Eds., xe2x80x9cNeurotransmitters and Epilepsyxe2x80x9d, Wiley-Liss, New York, N.Y., 1991) and stroke (Meldrum, B. S. and Garthwaite, J., TIPS, 11, 379-385, 1990; Rothman, S. M. and Olney, J. W., Ann. Neurol., 19, 105-111, 1986). Both diseases have been suggested to have a common pathology, i.e., chronic or acute cell death resulting from EAA-induced xe2x80x9cexcitotoxicityxe2x80x9d (Greenamyre, J. T., Maragos, W. F., Albin, R. L., Penny, J. B., and Young, A. B., Prog. Neuro Psychopharmacol and Biol. Psychiat., 12, 421, 1988; Mayer, M. L., and Westbrook, G. L., Prog. Neurobiol., 28, 197, 1987; Choi, D. S., Neuron, 1, 623, 1988; Simpson, M. D. C., Royston, M. C., Deakin, J. F. W., Cross, A. J., Mann, D. M. A., and Slater, P., Brain Res., 462, 76, 1988). Excessive accumulation of glutamate leads to overactivation of the NMDA receptor resulting in excessive intraneuronal Ca2+ which precipitates neurodegeneration and neuronal death (Cotman, C. W., Bridges, R. J., Taube, J. S., Clark, A. S., Geddes, J. W., and Monaghan, D. T., J. NIH Res., 1, 65, 1989). Evidence for the excitotoxic action of glutamate at the NMDA receptor, derived from numerous studies of cultured cortical neurons in vitro (Choi, D. S., Neuron, 1, 623, 1988), suggests an influx of Ca2+ through the stimulated NMDA ionophore to be a prerequisite for cell death to occur (Choi, D. S., Neuron, 1, 623, 1988; Hahn, J. S., Aizenman, E., and Lipton, S. A., Proc. Natl. Acad. Sci., 85, 6556, 1988; Ogura, A., Miyamoto, M. and Kudo, Y., Exp. Brain Res. 73, 447, 1988). Agents that block the action of glutamate and thus the overstimulation of the NMDA receptor thus represent novel therapies, as neuroprotective agents, for both epilepsy and cerebral ischemia resulting from stroke (Johnson, G., Ann. Rep. Med. Chem., 24, 41, 1989; Cotman, C. W., Bridges, R. J., Taube, J. S., Clark, A. S., Geddes, J. W., and Monaghan, D. T., J. NIH Res., 1, 65, 1989). Thus, based on the ability of the triazoline and the AAP anticonvulsants to effectively impair glutamate neurotransmission, it appears logical to expect that these compounds would provide beneficial drug candidates for stroke-related ischemic brain damage.
EAAs and the Kindling Model of Epilepsy:
The kindling phenomenon mimics human epilepsy (Kalichman, M. W., Neurosci. Biobehav. Rev., 6, 165, 1982) and there is increasing evidence that EAAs may play an important role in kindling mechanisms. EAAs may be critically involved in both epileptogenesis and as a focus for the mechanism of action of anticonvulsants (Meldrum, B. S., and Chapman, A. G., In xe2x80x9cGlutamine, Glutamate, and GABA in the Central Nervous System,xe2x80x9d, L. Hertz, et al., Ed., Alan R. Liss, Inc., New York, 1983, pp 625-641; Cavalheiro, E. A., Lehmann, J., and Turski, L., Eds., xe2x80x9cFrontiers in Excitatory Amino Acid Researchxe2x80x9d, A. R. Liss, New York, N.Y., 1988; Muir, K. W. and Lees, K. R., Stroke, 26, 503-513, 1995). Enhanced activity at the EAA synapse will lower the threshold and promote hyperactivity of the postsynaptic neuron. Evidence for a causal connection between EAA release and onset of hyperactivity has been provided by the use of specific EAA receptor antagonists, APB, APV, and APH, in various models of epilepsy (Cruczwar, S. J., and Meldrum, B. S., Eur. J. Pharmacol., 83, 335, 1982).
EAAs and Cerebral Ischemia:
Brain regions such as the hippocampus and the dorsolateral striatum that are enriched in EAA receptors are especially vulnerable to ischemic lesions (Jorgensen, M. D. and Diemer, N. A., Acta Neurol. Scand., 66, 536-46, 1982) and selective brain lesioning studies have supported a role for glutamate in ischemic and hypoglycemic brain injury (Jorgensen, M. B., Johnson, F. F., and Diemer, N. H., Acta Neuropathol., 73, 189, 1987; Linden, T., Kalimo, H., and Weiloch, T., Acta Neuropathol., 74, 335, 1988). Furthermore, ischemia-induced hippocampal damage is reduced by prior local infusion of EAA receptor antagonists (Simon, R. P., Griffiths, T., Evans, M. C., Swan, J. H., and Meldrum, B. S., J. Cereb. Blood Flow Metab., 4, 350-361, 1984; Simon, R. P., Swan, J. H., Griffiths, T., and Meldrum, B. S., Science, 226, 850-852, 1984) or by their systemic administration (Boast, C. A., Gerhardt, S. C., Pastor, G., Lelunann, J., Etienne, P. E., and Liebman, J. M., Brain Res., 442, 345-348, 1988). Glutamate can trigger toxic neuronal degeneration with considerable potency and speed; a 5-minute exposure to 100 xcexcM Glu is sufficient to destroy large numbers of cultured cortical neurons (Choi, D. W., Maulucci-Gedde, M. A., Kriegstein, A. R., J. Neurosci., 7, 357-368, 1987). Such brief intense exposure likely accompanies several types of acute insults, including hypoxia (Rothman, S. M., J. Neurosci., 4, 188-191, 1984), ischemia (Simon, R. P., Griffiths, T., Evans, M. C., Swan, J. H., and Meldrum, B. S., J. Cereb. Blood Flow Metab., 4, 350-361, 1984; Simon, R. P., Swan, J. H., Griffiths, T., and Meldrum, B. S., Science, 226, 850-852, 1984) and prolonged seizures (Ben-Ari, Y., Neuroscience, 14, 375-403, 1985).
In the hippocampus, the pattern of neuronal loss is similar after an episode of ischemia or of status epilepticus or temporal lobe epilepsy, the most common form of focal (partial) epilepsy. Irreversible cell loss is common in the hilus of the hippocampal area dentata and in the CA1 and CA3 pyramidal cell layers. Prolonged (24 hours) electrical stimulation of the perforant path fibers in vivo produces histopathological changes in the hippocampal CA1 and CA3 pyramidal neurons similar to those elicited by EAAs (Meldrum, B. S., and Corsellis, J. A. N., In xe2x80x9cGreenfield""s Neuropathologyxe2x80x9d, 4th Edn., J. H. Adams, et al., Ed., 1984, pp 921-950; Sloviter, R. S., Brain Res. Bull., 10, 675-697, 1983; Sloviter, R. S., Science, 73, 1987). The increased activity in excitatory hippocampal pathways is suggested as the cause for the irreversible damages to cells, probably by the release of EAAs in neurotoxic concentrations followed by Ca2+ influx through the stimulated NMDA receptor-ion channel complex. The mitochondria in selectively vulnerable hippocampal neurons show massive overloading with Ca2+ during status epilepticus and after 2 hours of reperfusion following cerebral ischemia (Griffiths. T., Neuroscience, 10, 385-395, 1983).
The compounds of the present invention are useful in pharmaceutical compositions using conventional pharmaceutical carriers or vehicles for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, suppositories, sterile parenteral solutions or suspensions, sterile non-parenteral solutions or suspensions, oral solutions or suspensions, oil in water or water in oil emulsions and the like, containing suitable quantities of the active ingredient.
Compositions for injection, may be prepared in unit dosage form in ampules, or in multidose containers. The injectable compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain various formulating agents. Alternatively, the active ingredient may be in powder form for reconstitution at the time of delivery with a suitable vehicle, such as sterile water. In injectable compositions, the carrier is typically comprised of sterile water, saline or another injectable liquid. Also, various buffering agents, preservatives and the like can be included.
Topical applications may be formulated in carriers such as hydrophobic or hydrophilic bases to form ointments, creams, lotions, in aqueous, oleaginous or alcoholic liquids to form paints or in dry diluents to form powders.
Oral compositions may take such forms as tablets, capsules, oral suspensions and oral solutions. The oral compositions may utilize carriers such as conventional formulating agents, and may include sustained release properties as well as rapid delivery forms.
The dosage to be administered depends to a large extent upon the condition and size of the subject being treated, the route and frequency of administration, and the particular compound selected. Such matters, however, are left to the routine discretion of the physician according to principles of treatment well known in the medical arts. The compositions of this invention for human delivery per unit dosage, whether liquid or solid, comprise from about 0.01% to as high as about 99% of the active compound, the preferred range being from about 10-60%.
The invention described herein also includes a method of treating a mammal in need of ischemia treatment comprising administering to said mammal the claimed composition in an amount effective to treat said condition. About 1 to 300 mg/kg of body weight, preferably about 25 to 200 mg/kg, one to four times daily is preferred.
The 5-pyridyl substituted triazoline compounds represented by structures I, II and III of this invention may be prepared by the reaction of diazomethane with Schiff bases as described in the Applicant""s previous patents on triazolines (P. K. Kadaba, U.S. Pat. No. 4,511,572, 1985; U.S. Pat. No. 4,689,334, 1987, the disclosures of which are hereby incorporated by reference), and illustrated in Equation 1. 
where R2 is 4-, 3- or 2-pyridyl and R1 is 3,4- or 3,5-dichloro, p- or m-chloro, p- or m-bromo, p- or m-fluoro, p- or m-trifluoromethyl, p- or m-lower alkyl, p- or m-lower alkoxy or hydrogen.
In the method of preparation, the reaction between the Schiff base and diazomethane is carried out by treating the appropriate Schiff base with a dioxane solution of diazomethane at room temperature, as described previously (P. K. Kadaba, U.S. Pat. No. 4,511,572, 1985; U.S. Pat. No. 4,689,334, 1987).
The following examples are presented to illustrate the invention, but it is not to be considered as limited thereto. In the examples and throughout the specifications, parts are by weight unless otherwise indicated.